Advances in recombinant DNA technology in recent years have made possible the production of significant quantities of foreign proteins of interest in host cells. Recombinant proteins are produced in host cell systems by transfecting the host cells with DNA coding for the protein of interest, and then growing the transfected host cells under conditions which favor expression of the new recombinant protein by the host cell. Where the recombinant protein of interest is highly expressed by a particular host cell system, these exogenous proteins are typically precipitated within the host cell as inclusion bodies. High levels of expression, and consequent deposition of the recombinant protein in the form of inclusion bodies, is more likely to occur where procaryotic host cells are employed.
The procaryote E. coli is commonly selected for use in high expression systems, in part, because E. coli host cells tend to be more amenable to the production of extremely large quantities of recombinant protein. Low expression host cell systems, typically those employing eucaryotic host cells and yeast host cells, fail to produce recombinant protein in the tremendous quantities generated in high expression host cell systems. While expressed in relatively low quantities, however, recombinant proteins from these lower expression host cells are more likely to be recovered in their biologically active form, due to the tendency of low expression host cells to secrete the exogenous recombinant protein into the aqueous medium surrounding the host cell, rather than to deposit the protein in the high density inclusion bodies.
The trade-off with higher expression systems is that, in return for obtaining higher yields of recombinant product, the recombinant protein must be isolated from inclusion bodies. This typically requires refolding of the denatured protein in order to generate biologically active product. Both the difficulty and the success of efforts to refold recombinant proteins varies significantly with the particular protein being produced.
One recombinant protein which has become of particular interest in the wake of recent advances in recombinant DNA technology is a growth factor known as insulin-like growth factor I (IGF-I). IGF-I is known to consist of 70 amino acids, as is shown in the following sequence: ##STR1##
IGF is biologically active only in its refolded form. The IGF-I molecule contains six cysteine residues, all of which form disulfide bonds which hold the molecule in its biologically active refolded form.
The difficulty inherent in obtaining biologically active recombinant proteins from high expression host cell systems is particularly difficult in the case of IGF-I due to the tendency of the IGF-I molecule to form stable, but incorrect disulfide bonds, which result in inactive, or only partially active, conformers. Moreover, typical methods of refolding IGF-I may result in a mixture of these conformers, which are then very difficult to separate. Procaryotic hosts thus have not been found to produce active recombinant IGF-I (rIGF-I) in significant yields (e.g., gram quantities) due to the difficulty in refolding IGF-I and the difficult separation problems.
With respect to recombinant proteins in general, however, refolding methods have been used for transforming denatured recombinant proteins into their active form. U.S. Pat. Nos. 4,511,503 and 4,518,256, for example, describe three refolding procedures which are regarded as being universally applicable, with only minor modifications, to the recovery of biologically active recombinant proteins from inclusion bodies. These procedures recognize that the tertiary, or refolded, structure of proteins is stabilized by hydrogen bonding, hydrophobic interactions, and ionic bonding between amino acid moieties of the protein. When present, it is the disulfide bonding between cysteine moieties that "locks" the tertiary structure in place. These methods therefore seek to eliminate random disulfide bonding prior to coaxing the recombinant protein into its biologically active conformation through its other stabilizing forces.
In one approach, the denatured protein of interest is further purified, under reducing conditions which maintain the cysteine moieties of the protein as free sulfhydryl groups, by supplying a reducing agent throughout all of the purification steps. This permits the protein to refold itself under the conditions of purification, in the absence of incorrect disulfide bond formation. The reducing agent is then diluted into an aqueous solution to enable the refolded protein to form the appropriate disulfide bonds in the presence of air or some other oxidizing agent. This enables refolding to be easily incorporated into the overall purification process. This method works best for recombinant proteins which have relatively uncomplicated tertiary structures in their biologically active forms.
In another approach, refolding of the recombinant protein is allowed to occur in the presence of both the reduced (R-SH) and oxidized (R-S-S-R) forms of a sulfhydryl compound. This enables free sulfhydryls and disulfides to be constantly formed and reformed throughout the purification process. The reduced and oxidized forms of the sulfhydryl compound are provided in a buffer having sufficient denaturing power that all of the intermediate conformations of the protein remain soluble in the course of the unfolding and refolding. Urea is suggested as a suitable buffer medium because of its apparent ability to act as both: (1) a weak enough denaturing agent to allow the protein to approximate its correct conformation; and, (2) a strong enough denaturant that the refolding intermediates maintain their solubility. This approach also works best where the recombinant inclusion body proteins of interest have relatively uncomplicated folding patterns.
A third approach, which is used in more difficult refolding situations, is designed to first break any disulfide bonds which may have formed incorrectly during isolation of the inclusion bodies, and then to derivatize the available free sulfhydryl groups of the recombinant protein. This is accomplished by sulfonating the protein to form a protein-S-SO.sub.3 bond. The resulting protein-S-sulfonate solution is then diluted into an aqueous solution where proper refolding is allowed to occur in the absence of incorrect disulfide bond formation. A system containing a sulfhydryl compound (R-SH) and a small percentage of its corresponding oxidized form (R-S-S-R), is then added to the aqueous solution. The pH is adjusted (raised) to a value such that the sulfhydryl compound (R-SH) is at least partially in ionized form (R-S-) so that nucleophilic displacement of the sulfonate is enhanced. While the sulfhydryl compound is sufficient to effect conversion of the protein-S-sulfonate to the appropriate disulfide binding partner, the presence of an oxidized form is required to insure that suitable disulfide bonds will remain intact.
These refolding methods have not been shown to work efficiently with rIGF-I and/or are burdensome and expensive to perform.
In addition, methods have been suggested for preparing IGF-I as a fusion protein. For example, European Patent Application No. 219,814 discloses a process for preparing IGF-I fused with a "protective peptide". These methods employing fusion proteins, however, generally require a relatively long leader sequence, and are directed to improving expression of the inclusion body protein, not to improving refolding of the denatured recombinant protein.
Accordingly, it is an object of the present invention to provide a method for the isolation and purification of biologically active rIGF-I from high expression host cell systems.